Patent Application
20210062160
This invention generally relates to medicine, infectious diseases, immunology, pharmacology and microbiology. In alternative embodiments, provided are compositions and methods for treating, ameliorating and preventing various infections, disorders and conditions in mammals, including: delivering: (i) a bacteriophage (“phage”), wherein optionally the phage is a temperate phage or a lysogenic phage (ii) a prophage (optionally a tailocin, or a defective prophage where head and tail are absent but the prophage is otherwise adsorption-competent), a phagemid or a phage-like particle (optionally a phagocin), (iii) a general transducing agent (GTA), or a small, tailed bacteriophage-like particle, (iv) a Metamorphosis Associated Contractile structure (MACs), (v) a phage-derived product (optionally an endolysin, a holin, a lysozyme, or a tail fiber protein), into the blood stream or lymphatic system of an animal, e.g., a mammal, or delivering the bacteriophage, phagemid or phage-like particle, etc to a tissue or organ of the animal in vivo; treating, ameliorating and/or preventing a bacterial or viral infection in the animal in vivo, wherein the bacterial or viral infection in the animal is outside of the gut of the mammal, wherein optionally the bacterial or viral infection comprises a lung infection, or a secondary infection outside of the gut; generating an immune response in the animal by delivering the bacteriophage, phagemid or phage-like particle, etc into the blood stream or lymphatic system of the mammal, wherein optionally the immune response is a humoral (antibody) response, a cell-mediated immune response, or a tolerogenic immune (suppressing) response; treating, ameliorating and/or preventing a disease or condition in an individual in need thereof, wherein optionally the disease or condition comprises obesity, diabetes, autism, a cystic fibrosis, an inflammation outside of the gut; and/or delivering a payload or a composition in vivo to the animal, or labelling, tagging or coating a cell in vivo in the animal, comprising administering or applying to the animal in vivo, or to an individual in need thereof: a composition, a product of manufacture, a food, a drink, a nutraceutical, a formulation, a pharmaceutical or a pharmaceutical preparation comprising the bacteriophage, phagemid or phage-like particle, etc, which optionally comprise a payload, e.g., a drug, an effector nucleic acid, an immunogen, a label.
Bacteriophages can freely and profusely penetrate our bodies and the bodies of other higher vertebrates (4, 5)(6, 7). Phages have been detected in the blood and serum of both symptomatic and asymptomatic humans (8-12). Dosing phages to mice via oral feeding and gastric lavage resulted in the rapid migration of phage into the blood stream that was both irregular but repeatable (6). Phage migration to the blood was rapidly followed by their permeation into all major organs of the body, including the lung, liver, kidney, spleen, urinary tract and even the brain, indicating their capacity to cross the blood-brain barrier (6, 13-16).
Within the human body the largest reservoir of phages is within the gut (17, 18). From here there are several possible routes by which gut phages could penetrate the body. The most rudimentarily proposed route of access is via a ‘leaky gut’, characterized by cellular damage and punctured vasculature at sites of inflammation, allowing phages to bypass confluent epithelial layers (19, 20). Other proposed mechanisms include; ‘trojan horse’ whereby phages infect a bacterium, which then enters or is engulfed by an epithelial cells (21-23), ‘phage display’ a process that requires homing ligands to be displayed onto viral coats for cellular recognition and receptor-mediated transcytosis (24-28), and the ‘free uptake’ of phage particles by eukaryotic cells via endocytosis (22, 29, 30). There is supporting and contrasting evidence for all of these mechanisms, suggesting that phages may access the body via diverse routes. Few attempts have been made to investigate whether phage transcytosis occurs naturally and, consequently the primary route that phages use to access the body has yet to be identified.
In alternative embodiments, provided are methods for:
delivering: (i) a bacteriophage (“phage”), wherein optionally the phage is a temperate phage or a lysogenic phage (ii) a prophage (optionally a tailocin, or a defective prophage where head and tail are absent but the prophage is otherwise adsorption-competent), a phagemid or a phage-like particle (optionally a phagocin), (iii) a general transducing agent (GTA), or a small, tailed bacteriophage-like particle, (iv) a Metamorphosis Associated Contractile structure (MACs), (v) a phage-derived product (optionally an endolysin, a holin, a lysozyme, or a tail fiber protein), or (vi) any combination of (i) to (v), into an animal (optionally a mammal or a human),
delivering: (i) a bacteriophage (“phage”), wherein optionally the phage is a temperate phage or a lysogenic phage (ii) a prophage (optionally a tailocin, or a defective prophage where head and tail are absent but the prophage is otherwise adsorption-competent), a phagemid or a phage-like particle (optionally a phagocin), (iii) a general transducing agent (GTA), or a small, tailed bacteriophage-like particle, (iv) a Metamorphosis Associated Contractile structure (MACs), (v) a phage-derived product (optionally an endolysin, a holin, a lysozyme, or a tail fiber protein), or (vi) any combination of (i) to (v), into a eukaryotic cell,
treating, ameliorating and/or preventing a bacterial or viral infection in an animal in vivo, wherein optionally the bacterial or viral infection in the animal is inside or outside of the gut of the animal,
generating or modulating an immune response in an animal by delivering: (i) a bacteriophage (“phage”), wherein optionally the phage is a temperate phage or a lysogenic phage (ii) a prophage (optionally a tailocin, or a defective prophage where head and tail are absent but the prophage is otherwise adsorption-competent), a phagemid or a phage-like particle (optionally a phagocin), (iii) a general transducing agent (GTA), or a small, tailed bacteriophage-like particle, (iv) a Metamorphosis Associated Contractile structure (MACs), (v) a phage-derived product (optionally an endolysin, a holin, a lysozyme, or a tail fiber protein), or (vi) any combination of (i) to (v), into the animal (optionally a mammal or a human),
treating, ameliorating and/or preventing a disease or condition in an individual in need thereof,
delivering a payload or a composition in vivo to an animal, or labelling, tagging or coating a cell in vivo in an animal,
administering or applying: to the animal, optionally in vivo, or to the individual in need thereof; or, or administering or applying or inserting into or onto the eukaryotic cell:
wherein optionally the (i) the bacteriophage (“phage”) (ii) the prophage, the phagemid or the phage-like particle, (iii) the general transducing agent (GTA), or the small, tailed bacteriophage-like particle, (iv) the Metamorphosis Associated Contractile structure (MACs), (v) the phage-derived product, or (vi) any combination of (i) to (v) is: chemically or structurally modified, genetically engineered, or is a synthetic version or construct,
and optionally the (i) the bacteriophage (“phage”) (ii) the prophage, the phagemid or the phage-like particle, (iii) the general transducing agent (GTA), or the small, tailed bacteriophage-like particle, (iv) the Metamorphosis Associated Contractile structure (MACs), (v) the phage-derived product, or (vi) any combination of (i) to (v), comprises or has contained thereon or within a payload, wherein optionally the payload comprises a composition heterologous to (i) the bacteriophage (“phage”) (ii) the prophage, the phagemid or the phage-like particle, (iii) the general transducing agent (GTA), or the small, tailed bacteriophage-like particle, (iv) the Metamorphosis Associated Contractile structure (MACs), (v) the phage-derived product,
and optionally the heterologous composition is capable of treating, ameliorating and/or preventing a disease or condition in the individual in need thereof, or repairing a defect in the eukaryotic cell, or adding or modifying a function in the eukaryotic cell, or altering the genome of or a nucleic acid in the eukaryotic cell,
and optionally the (i) the bacteriophage (“phage”) (ii) the prophage, the phagemid or the phage-like particle, (iii) the general transducing agent (GTA), or the small, tailed bacteriophage-like particle, (iv) the Metamorphosis Associated Contractile structure (MACs), (v) the phage-derived product, or (vi) any combination of (i) to (v), has a size ranging from between about 1 nm and 1000 nm, or between about 100 and 500 nm, or between about 1 nm and 10 μm.
In alternative embodiments, the individual is a mammal or a human, and optionally the mammal is a human, a human infant, and optionally the animal is wildlife, livestock, beef, poultry, or a domesticated or a laboratory animal.
In alternative embodiments, an antacid or a buffer or buffering agent or a pharmaceutically acceptable excipient is administered before, during or after, or before and during, administration of the composition, product of manufacture, food, drink, nutraceutical, formulation, pharmaceutical or pharmaceutical preparation.
In alternative embodiments, a sufficient amount of antacid, buffer or buffering agent is administered (optionally before, during or after, or before and during, administration) to raise the pH of the stomach in the individual to between about 2.5 and 7, or between about 3 and 6.5, or to about 5.0, 5.5, 6.0, 6.5, 6.8 or 7.0 (optionally these pH values reached before, during or after, or before and during, administration), and optionally the buffer or a buffering agent or the pharmaceutically acceptable excipient comprises an inorganic salt, a citric acid, a sodium chloride, a potassium chloride, a sodium sulfate, a potassium nitrate, a sodium phosphate monobasic, a sodium phosphate dibasic or combinations thereof, and optionally the antacid comprises a calcium carbonate, a magnesium hydroxide, a magnesium oxide, a magnesium carbonate, an aluminum hydroxide, a sodium bicarbonate or a dihydroxyaluminum sodium carbonate.
In alternative embodiments, the (i) bacteriophage (“phage”) (ii) prophage, the phagemid or phage-like particle, (iii) general transducing agent (GTA), or small, tailed bacteriophage-like particle, (iv) Metamorphosis Associated Contractile structure (MACs), (v) phage-derived product, or (vi) any combination of (i) to (v), is capable of specifically binding to an animal (optionally a mammalian or a human cell), or is capable of specifically binding to a specific animal cell, and optionally the (i) bacteriophage (“phage”) (ii) prophage, the phagemid or phage-like particle, (iii) general transducing agent (GTA), or small, tailed bacteriophage-like particle, (iv) Metamorphosis Associated Contractile structure (MACs), (v) phage-derived product, or (vi) any combination of (i) to (v), is engineered to target a specific cell, tissue or organ, or diseased, infected or abnormal cell.
In alternative embodiments, an immune response is generated by display of epitopes or immunogens, or tolerogens, or immune response modulators, on the surface of the delivered or administered: (i) bacteriophage (“phage”) (ii) prophage, the phagemid or phage-like particle, (iii) general transducing agent (GTA), or small, tailed bacteriophage-like particle, (iv) Metamorphosis Associated Contractile structure (MACs), (v) phage-derived product, or (vi) any combination of (i) to (v), or by the inclusion of epitopes or immunogens, or tolerogens, or immune response modulators in the delivered or administered: (i) bacteriophage (“phage”) (ii) prophage, the phagemid or phage-like particle, (iii) general transducing agent (GTA), or small, tailed bacteriophage-like particle, (iv) Metamorphosis Associated Contractile structure (MACs), (v) phage-derived product, or (vi) any combination of (i) to (v).
In alternative embodiments, the (i) bacteriophage (“phage”) (ii) prophage, the phagemid or phage-like particle, (iii) general transducing agent (GTA), or small, tailed bacteriophage-like particle, (iv) Metamorphosis Associated Contractile structure (MACs), (v) phage-derived product, or (vi) any combination of (i) to (v), is/are formulated per dose, or per serving, or per unit dosage at, or at a total daily dose of: between about 10(1)(or 101) and 10(20) plaque-forming units (PFUs), or between about 10(3) and 10(17) PFUs, or between about 10(5) and 10(12) PFUs, or between about 10(7) and 10(9) PFUs.
In alternative embodiments, the (i) bacteriophage (“phage”) (ii) prophage, the phagemid or phage-like particle, (iii) general transducing agent (GTA), or small, tailed bacteriophage-like particle, (iv) Metamorphosis Associated Contractile structure (MACs), (v) phage-derived product, or (vi) any combination of (i) to (v), comprises, or contains within or upon, or carries, a payload or a composition,
wherein optionally the composition or the payload comprises: a drug; a modulator of transcytosis, endocytosis, exocytosis, receptor mediated endocytosis, non-specific binding, pinocytosis or macrocytosis in a eukaryotic cell; an immune response modulator, a epitope, an immunogen or a tolerogen; an antibiotic or a bacteriostatic agent; a cytotoxic agent; a nucleic acid (optionally an RNA (optionally an iRNA or miRNA), or an antisense nucleic acid, or a ribozyme, or a CRISPR or CRISPR/Cas9 nucleic acid, or a CRISPR/Cas9-gRNA complex for genome editing, or a DNA), wherein optionally the nucleic acid is derived from a phage, a bacterial or an animal, and optionally the nucleic acid is a synthetic or a recombinantly engineered nucleic acid, optionally the nucleic acid comprises a eukaryotic gene with the appropriate regulatory motifs, optionally promoters, such that the gene is expressed in a eukaryotic cell, optionally a gut cell; a genome or fragment thereof, wherein optionally the genome is derived from a phage or a bacterial genome; a carbohydrate, a protein or peptide, a lipid, an antibody or a small molecule; a label or tag or a fluorescent molecule or a radiopaque molecule; a magnetic particle; a radionucleotide; a carbohydrate binding domain (CBD) or a moiety or domain capable of binding to: a protein or peptide, a nucleic acid (optionally an RNA or a DNA), a lipid, a lipo-polysaccharide or a mucopolysaccharide; or, any combination thereof, wherein optionally the modulator of transcytosis, endocytosis, exocytosis, receptor mediated endocytosis, non-specific binding, pinocytosis or macrocytosis in the eukaryotic cell comprises or is an inhibitor or enhancer of transcytosis, endocytosis, exocytosis, receptor mediated endocytosis, non-specific binding, pinocytosis or macrocytosis, and optionally the inhibitor of transcytosis, endocytosis, exocytosis, receptor mediated endocytosis, non-specific binding, pinocytosis or macrocytosis is or comprises N-ethylmaleimide (NEM), chlorpromazine, filipin, colchicine, dynasore, Concanamycin C (Con C), eeyarestatin I, Golgicide A. Leptomycin B, levetiracetam, or brefeldin A (BFA), or an antibody that inhibits PIKFyve or a SNARE protein or an antibody that blocks SNARE assembly,
and optionally the nucleic acid is or comprises a small inhibitory RNA (siRNA), an antisense nucleic acid or RNA, or a CRISPR nucleic acid or CRISPR/Cas9 system comprising a synthetic guide RNA (gRNA) and/or a nuclease, or the nucleic acid encodes a protein or a small inhibitory RNA (siRNA), an antisense RNA, or a CRISPR nucleic acid or CRISPR/Cas9 system comprising a synthetic guide RNA (gRNA) and/or a nuclease,
and optionally the nucleic acid is contained in an expression vehicle or vector, and optionally the nucleic acid is operatively linked to a transcriptional control motif, which optionally can be a promoter and/or enhancer, optionally a tissue or cell specific, or constitutive, or inducible, promoter and/or enhancer,
and optionally the payload or composition is delivered to or released in, onto or into the eukaryotic cell, or is delivered or released into a eukaryotic cell subcellular compartment or an organelle,
and optionally the eukaryotic cell subcellular compartment or organelle is a cytoplasm, an endosome, an exosome, a liposome, a nucleus, a nucleosome, a golgi, an endoplasmic reticulum (ER) or a mitochondria,
and optionally the (i) bacteriophage (“phage”) (ii) prophage, the phagemid or phage-like particle, (iii) general transducing agent (GTA), or small, tailed bacteriophage-like particle, (iv) Metamorphosis Associated Contractile structure (MACs), (v) phage-derived product, or (vi) any combination of (i) to (v), is engineered to release the payload or composition into the eukaryotic cell, or eukaryotic cell subcellular compartment or organelle, or into a specific eukaryotic cell subcellular compartment or organelle,
and optionally the (i) bacteriophage (“phage”) (ii) prophage, the phagemid or phage-like particle, (iii) general transducing agent (GTA), or small, tailed bacteriophage-like particle, (iv) Metamorphosis Associated Contractile structure (MACs), (v) phage-derived product, or (vi) any combination of (i) to (v), is degraded in a lysosome, or is engineered or designed to be degraded in a lysosome.
In alternative embodiments, the (i) bacteriophage (“phage”) (ii) prophage, the phagemid or phage-like particle, (iii) general transducing agent (GTA), or small, tailed bacteriophage-like particle, (iv) Metamorphosis Associated Contractile structure (MACs), (v) phage-derived product, or (vi) any combination of (i) to (v), is or is derived from, or is substantially or partially derived from:
(a) a prokaryotic bacteriophage, optionally a bacterial or an Archaeal bacteriophage;
(b) a prokaryotic bacteriophage of the order Caudovirales or Ligamenvirales;
(c) a prokaryotic bacteriophage of the family Myoviridae, Siphoviridae, Podoviridae, Lipothrixviridae, Rudiviridae, Ampullaviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Cystoviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Inoviridae, Leviviridae, Microviridae, Plasmaviridae or Tectivirus or a combination thereof;
(d) a Bacteroidetes-infecting phage or a class I filamentous phage, or an F1 or an Fd filamentous bacteriophage:
(e) a bacteriophage Qβ virus-like particle; or
(f) an Enterobacteria phage T4, a lambda phage, an M13 Inoviridae phage, a crAss phage, or a phage capable of infecting a mammalian or a human gut.
In alternative embodiments, the (i) bacteriophage (“phage”) (ii) prophage, the phagemid or phage-like particle, (iii) general transducing agent (GTA), or small, tailed bacteriophage-like particle, (iv) Metamorphosis Associated Contractile structure (MACs), (v) phage-derived product, or (vi) any combination of (i) to (v), is a chemically or structurally modified bacteriophage, phagemid or phage-like particle, and optionally the exterior (outer) surface of bacteriophage, phagemid or phage-like particle comprises:
(a) at least one heterologous:
wherein optionally the heterologous CBD is a bacteriophage carbohydrate binding domain (CBD), and optionally the heterologous CBD is a CBD derived from a different species, genus, family or order of bacteriophage; or the CBD is a mammalian or a human CBD,
and optionally any of (i) to (iii) comprises or has structural homology to: a C-type lectin, a lectin, a bacteriodetes-associated carbohydrate-binding often N-terminal (BACON) domain, a Brefeldin A-inhibited guanine nucleotide-exchange factor for ADP-ribosylation factor (Big, optionally Big1, Big2, or Big3), a polycystic kidney disease domain (PKD), a Fibronectin type 3 homology domain (Fn3), a HYalin Repeat (HYR) domain, an Ig_2 domain, an immunoglobulin I-set domain, a carbohydrate-adherence domain, a mucus-binding protein, a glycan-binding protein, a protein-binding protein, a mucus-adhering protein or a mucus-adhering glycoprotein:
(b) additional homologous CBDs (more CBDs than found on a comparable wild type (WT) bacteriophage); or
(c) a combination of (a) and (b).
In alternative embodiments, the (i) bacteriophage (“phage”) (ii) prophage, the phagemid or phage-like particle, (iii) general transducing agent (GTA), or small, tailed bacteriophage-like particle, (iv) Metamorphosis Associated Contractile structure (MACs), (v) phage-derived product, or (vi) any combination of (i) to (v), comprises or has contained therein a genome (optionally a substantially complete or a partial, or a genetically engineered or hybrid genome) that is altered such that after reproduction in a host cell (optionally a bacterial host cell), or in an in vitro system, the exterior (outer) surface of the bacteriophage comprises:
(a) at least one non-bacteriophage carbohydrate binding domain (CBD), and optionally the CBD is a mammalian or a human CBD;
(b) at least one heterologous bacteriophage CBD, wherein optionally the heterologous CBD is a CBD from a different species, genus, family or order of bacteriophage:
(c) more CBDs than found on a wild type (WT) (comparable) bacteriophage; or
(d) at least one moiety or domain capable of binding to a component of a mucus,
(e) at least one moiety or domain capable of binding to a protein or peptide, a protein or peptide (optionally an antibody or antigen binding fragment thereof, an antigen, an immunogen, a tolerogen), a glycoprotein, a nucleic acid (optionally an RNA or a DNA), a lipid or cholesterol, a lipopolysaccharide, a mucopolysaccharide, a gel, a hydrogel, a complex fluid, or a combination thereof; or
(f) any combination of (a) to (e),
and optionally any of (a) to (e) comprises or has structural homology to: a C-type lectin, a lectin, a bacteriodetes-associated carbohydrate-binding often N-terminal (BACON) domain, a Brefeldin A-inhibited guanine nucleotide-exchange factor for ADP-ribosylation factor (Big, optionally Big1, Big2, or Big3), a polycystic kidney disease domain (PKD), a Fibronectin type 3 homology domain (Fn3), a HYalin Repeat (HYR) domain, an Ig_2 domain, an immunoglobulin I-set domain, a carbohydrate-adherence domain, a mucus-binding protein, a glycan-binding protein, a protein-binding protein, a mucus-adhering protein or a mucus-adhering glycoprotein.
In alternative embodiments:
(a) the CBD is entirely, or substantially, a synthetic or non-natural CBD, optionally an antibody or antigen binding domain that specifically binds to a carbohydrate:
(b) the CBD is or comprises a protein having a carbohydrate-binding-like fold, which optionally comprises a seven-stranded beta-sandwich, or optionally is or comprises an immunoglobulin-like binding domain, or a protein domain comprising a 2-layer sandwich of between 7 and 9 antiparallel Î2-strands arranged in two Î2-sheets; (c) the CBD is or is derived from or has substantial structural identity (homology) to a mammalian or a human CBD;
(d) the bacteriophage is known or demonstrated to be toxic or lysogenic to a bacteria, or the bacteriophage is bactericidal or bacteriostatic, or the bacteriophage can treat, inhibit or prevent an infection, and optionally the bacteriophage is engineered to specifically bind to or target the bacteria,
wherein optionally the bacteriophages are bactericidal or bacteriostatic to a gram negative bacteria or a gram positive bacteria, and optionally the bacteriophage is engineered to specifically bind to or target the gram negative bacteria or gram positive bacteria,
and optionally the bacteria or infection is or is caused by an MSRA infection, a Staphylococcus, a Staphylococcus aureus, a Clostridium, a Clostridium difficile, a Escherichia coli, a Shigella, a Salmonella, a Campylobacter, a Chloerae, a Bacillus, a Yersinia or a combination thereof, and optionally the bacteriophage is engineered to specifically bind to or target the bacteria or
(e) the bacteriophage is made or identified by a process comprising: screening a plurality of bacteriophages for bactericidal or bacteriostatic properties against a bacterium of interest, and selecting the bacteriophages having a lysogenic or a bactericidal or bacteriostatic activity.
In alternative embodiments, the CBD is, or is derived from, or has substantial structural identity (homology to):
(a) a protein having a carbohydrate-binding-like fold, which optionally comprises a seven-stranded beta-sandwich, or optionally is or comprises an immunoglobulin-like binding domain, or comprises a protein domain comprising a 2-layer sandwich of between 7 and 9 antiparallel Î2-strands arranged in two Î2-sheets;
(b) a CBD, optionally an antibody or antigen binding fragment thereof, capable of specifically binding to a tumor associated carbohydrate antigen (TACA); or
(c) a carbohydrate-binding module family 1 (CBM1);
a carbohydrate-binding module family 2 (CBM2);
a carbohydrate-binding module family 3 (CBM3);
a carbohydrate-binding module family 4 (CBM4);
a carbohydrate-binding module family 5 (CBM5);
a carbohydrate-binding module family 6 (CBM6):
a carbohydrate-binding module family 7 (CBM7);
a carbohydrate-binding module family 8 (CBM8);
a carbohydrate-binding module family 9 (CBM9);
a carbohydrate-binding module family 10 (CBM10);
a carbohydrate-binding module family 11 (CBM11);
a carbohydrate-binding module family 12 (CBM12);
a carbohydrate-binding module family 13 (CBM13);
a carbohydrate-binding module family 14 (CBM14);
a carbohydrate-binding module family 15 (CBM15);
a carbohydrate-binding module family 16 (CBM16);
a carbohydrate-binding module family 17 (CBM17);
a carbohydrate-binding module family 18 (CBM18);
a carbohydrate-binding module family 19 (CBM19);
a carbohydrate-binding module family 20 (CBM20);
a carbohydrate-binding module family 21 (CBM21);
a carbohydrate-binding module family 25 (CBM25);
a carbohydrate-binding module family 27 (CBM27);
a carbohydrate-binding module family 28 (CBM28);
a carbohydrate-binding module family 33 (CBM33);
a carbohydrate-binding module family 48 (CBM48); or,
a carbohydrate-binding module family 49 (CBM49).
In alternative embodiments, provided are uses of: a composition, a product of manufacture, a food, a drink, a nutraceutical, a formulation, a pharmaceutical or a pharmaceutical preparation, wherein the composition, product of manufacture, food, drink, nutraceutical, formulation, pharmaceutical or pharmaceutical preparation is or comprises a composition, product of manufacture, food, drink, nutraceutical, formulation, pharmaceutical or pharmaceutical preparation as used in a method of any of the preceding claims, in the preparation or manufacture of a medicament for:
delivering: (i) a bacteriophage (“phage”), wherein optionally the phage is a temperate phage or a lysogenic phage (ii) a prophage (optionally a tailocin, or a defective prophage where head and tail are absent but the prophage is otherwise adsorption-competent), a phagemid or a phage-like particle (optionally a phagocin), (iii) a general transducing agent (GTA), or a small, tailed bacteriophage-like particle, (iv) a Metamorphosis Associated Contractile structure (MACs), (v) a phage-derived product (optionally an endolysin, a holin, a lysozyme, or a tail fiber protein), or (vi) any combination of (i) to (v), into an animal (optionally a mammal or a human),
delivering: (i) a bacteriophage (“phage”), wherein optionally the phage is a temperate phage or a lysogenic phage (ii) a prophage (optionally a tailocin, or a defective prophage where head and tail are absent but the prophage is otherwise adsorption-competent), a phagemid or a phage-like particle (optionally a phagocin), (iii) a general transducing agent (GTA), or a small, tailed bacteriophage-like particle, (iv) a Metamorphosis Associated Contractile structure (MACs), (v) a phage-derived product (optionally an endolysin, a holin, a lysozyme, or a tail fiber protein), or (vi) any combination of (i) to (v), into a eukaryotic cell,
treating, ameliorating and/or preventing a bacterial or viral infection in an animal in vivo, wherein optionally the bacterial or viral infection in the animal is inside or outside of the gut of the animal,
generating or modulating an immune response in an animal by delivering: (i) a bacteriophage (“phage”), wherein optionally the phage is a temperate phage or a lysogenic phage (ii) a prophage (optionally a tailocin, or a defective prophage where head and tail are absent but the prophage is otherwise adsorption-competent), a phagemid or a phage-like particle (optionally a phagocin), (iii) a general transducing agent (GTA), or a small, tailed bacteriophage-like particle, (iv) a Metamorphosis Associated Contractile structure (MACs), (v) a phage-derived product (optionally an endolysin, a holin, a lysozyme, or a tail fiber protein), or (vi) any combination of (i) to (v), into the animal (optionally a mammal or a human),
treating, ameliorating and/or preventing a disease or condition in an individual in need thereof,
delivering a payload or a composition in vivo to an animal, or labelling, tagging or coating a cell in vivo in an animal,
and optionally the animal is a mammal or a human.
In alternative embodiments, provided are therapeutic formulations of a composition, a food, a drink, a nutraceutical, a formulation, a pharmaceutical or a pharmaceutical preparation,
wherein the composition, food, drink, nutraceutical, formulation, pharmaceutical or pharmaceutical preparation is or comprises a composition, food, drink, nutraceutical, formulation, pharmaceutical or pharmaceutical preparation as used in a method of any of the preceding claims,
for use in:
delivering: (i) a bacteriophage (“phage”), wherein optionally the phage is a temperate phage or a lysogenic phage (ii) a prophage (optionally a tailocin, or a defective prophage where head and tail are absent but the prophage is otherwise adsorption-competent), a phagemid or a phage-like particle (optionally a phagocin), (iii) a general transducing agent (GTA), or a small, tailed bacteriophage-like particle, (iv) a Metamorphosis Associated Contractile structure (MACs), (v) a phage-derived product (optionally an endolysin, a holin, a lysozyme, or a tail fiber protein), or (vi) any combination of (i) to (v), into an animal (optionally a mammal or a human),
delivering: (i) a bacteriophage (“phage”), wherein optionally the phage is a temperate phage or a lysogenic phage (ii) a prophage (optionally a tailocin, or a defective prophage where head and tail are absent but the prophage is otherwise adsorption-competent), a phagemid or a phage-like particle (optionally a phagocin), (iii) a general transducing agent (GTA), or a small, tailed bacteriophage-like particle, (iv) a Metamorphosis Associated Contractile structure (MACs), (v) a phage-derived product (optionally an endolysin, a holin, a lysozyme, or a tail fiber protein), or (vi) any combination of (i) to (v), into a eukaryotic cell,
treating, ameliorating and/or preventing a bacterial or viral infection in an animal in vivo, wherein optionally the bacterial or viral infection in the animal is inside or outside of the gut of the animal,
generating or modulating an immune response in an animal by delivering: (i) a bacteriophage (“phage”), wherein optionally the phage is a temperate phage or a lysogenic phage (ii) a prophage (optionally a tailocin, or a defective prophage where head and tail are absent but the prophage is otherwise adsorption-competent), a phagemid or a phage-like particle (optionally a phagocin), (iii) a general transducing agent (GTA), or a small, tailed bacteriophage-like particle, (iv) a Metamorphosis Associated Contractile structure (MACs), (v) a phage-derived product (optionally an endolysin, a holin, a lysozyme, or a tail fiber protein), or (vi) any combination of (i) to (v), into the animal (optionally a mammal or a human),
treating, ameliorating and/or preventing a disease or condition in an individual in need thereof,
delivering a payload or a composition in vivo to an animal, or labelling, tagging or coating a cell in vivo in an animal,
The details of one or more embodiments as provided herein are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.
The drawings set forth herein are illustrative of embodiments as provided herein and are not meant to limit the scope of the invention as encompassed by the claims.
as described in detail in Example 1, below.
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Like reference symbols in the various drawings indicate like elements.
Reference will now be made in detail to various exemplary embodiments of the invention, examples of which are illustrated in the accompanying drawings. The following detailed description is provided to give the reader a better understanding of certain details of aspects and embodiments of the invention, and should not be interpreted as a limitation on the scope of the invention.
In alternative embodiments, provided are compositions, products of manufacture and methods for treating, ameliorating and preventing various infections, disorders and conditions in animals, e.g., mammals such as humans, in vivo, including genetically-predisposed and chronic disorders, by administration to an individual in need thereof a composition, a product of manufacture, a food, a drink, a nutraceutical, a formulation, a pharmaceutical or a pharmaceutical preparation comprising: (i) a bacteriophage (“phage”), wherein optionally the phage is a temperate phage or a lysogenic phage (ii) a prophage (optionally a tailocin, or a defective prophage where head and tail are absent but the prophage is otherwise adsorption-competent), a phagemid or a phage-like particle (optionally a phagocin), (iii) a general transducing agent (GTA), or a small, tailed bacteriophage-like particle, (iv) a Metamorphosis Associated Contractile structure (MACs), (v) a phage-derived product (optionally an endolysin, a holin, a lysozyme, or a tail fiber protein), or (vi) any combination of (i) to (v), including chemically or structurally modified, genetically engineered and/or synthetic forms thereof; which optionally comprise a payload for having the desired effect, for example, the payload can be a drug, an effector nucleic acid, an immunogen, a label. In alternative embodiments, compositions, products of manufacture and methods as provided herein are effective for delivering payloads of any kind (e.g., drugs, small molecules, nucleic acids, immune response modulators, labels) to an animal cell, or an animal in vivo, to have a desired effect.
In alternative embodiments, compositions, products of manufacture and methods as provided herein are used to treat, prevent or ameliorate an infection in an animal, e.g., a mammal, in vivo inside or outside of the gastrointestinal tract (inside or outside of the gut). In alternative embodiment, compositions and methods as provided herein are used to specifically target and/or bind to an animal, e.g., mammalian, cell, optionally in vivo, that is associated with or completely or partially causative of an infection, disease or a condition.
In alternative embodiment, compositions, products of manufacture and methods as provided herein are designed to target a particular cell, tissue or organ, e.g., in vivo. In alternative embodiments, compositions, products of manufacture and methods as provided herein comprise use of (i) a bacteriophage (“phage”), (ii) a prophage, a phagemid or a phage-like particle, (iii) a general transducing agent (GTA), or a small, tailed bacteriophage-like particle, (iv) a Metamorphosis Associated Contractile structure (MACs), (v) a phage-derived product, or (vi) any combination of (i) to (v), to be specific for, or which are engineered, designed or constructed to be (e.g., by recombinant technology) specific for, or are capable of specifically targeting, a specific cell, tissue or organ in vivo, or a particular microbe, e.g., an infectious agent or a pathogen, or any microbe or bacteria that is pathogenic, or is associated with or completely or partially causative of an infection, disease or a condition.
In alternative embodiments, provided are compositions or products of manufacture, e.g., a drug delivery agent, a liposome or a micelle, a hydrogel, a dendrimer, a particle or a microparticle, a powder, a nanostructure or a nanoparticle, capable of targeting a specific cell, tissue or organ in vivo, or a microbe or bacteria, where in alternative embodiments the specific targeting is effected by incorporation of a component of (i) a bacteriophage (“phage”), (ii) a prophage, a phagemid or a phage-like particle, (iii) a general transducing agent (GTA), or a small, tailed bacteriophage-like particle, (iv) a Metamorphosis Associated Contractile structure (MACs), (v) a phage-derived product, or (vi) any combination of (i) to (v), which is designed or constructed to be (e.g., by recombinant technology) specific for, or responsible for or is capable of, specifically targeting, a specific cell, tissue or organ in vivo, or a specific microbe or bacteria, which can be a particular infectious agent or pathogen, a microbe or a bacteria that is pathogenic, or is associated with or completely or partially causative of an infection or a condition, for example, a bacteria.
Provided herein in Example 1, below, are data providing evidence demonstrating that (i) a bacteriophage (“phage”), (ii) a prophage, a phagemid or a phage-like particle, (iii) a general transducing agent (GTA), or a small, tailed bacteriophage-like particle, (iv) a Metamorphosis Associated Contractile structure (MACs), (v) a phage-derived product, or (vi) any combination of (i) to (v), as provided herein can: effectively and in sufficient amounts enter an animal, e.g., a mammal, e.g., an individual in need thereof, to deliver sufficient amounts of a payload, and optionally target to a specific cell, tissue or organ; or, enter into a tissue, the blood stream or a lymphatic system of the animal, e.g., a mammal, or to deliver a payload in vivo, for e.g., treating, ameliorating and/or preventing an infection, disease or condition in an individual in need thereof, where the infection, disease or condition is outside of the gut or gastrointestinal (GI) tract. Data evidence also demonstrates that phages, phagemids and phage-like particles and the like as provided herein can effectively and in sufficient amounts enter an animal, e.g., a mammal, to generate an immune response in the animal. In alternative embodiments, the immune response is generated by display of epitopes, tolerogens, drugs, or immunogens on the surface of, or within, the delivered (i) bacteriophage (“phage”) (ii) prophage, the phagemid or phage-like particle, (iii) general transducing agent (GTA), or small, tailed bacteriophage-like particle, (iv) Metamorphosis Associated Contractile structure (MACs), (v) phage-derived product, or (vi) any combination of (i) to (v).
In alternative embodiment, compositions, products of manufacture and methods as provided herein (e.g., comprising the (i) a bacteriophage (“phage”), (ii) a prophage, a phagemid or a phage-like particle, (iii) a general transducing agent (GTA), or a small, tailed bacteriophage-like particle, (iv) a Metamorphosis Associated Contractile structure (MACs), (v) a phage-derived product, or (vi) any combination of (i) to (v), as provided herein, optionally carrying a payload) target probiotic bacterial strains such that they can be engineered to constitutively produce phages and the like in the gut (or other organ or space) for delivery to e.g., epithelial cells.
In alternative embodiment, compositions, products of manufacture and methods as provided herein (e.g., comprising the (i) a bacteriophage (“phage”), (ii) a prophage, a phagemid or a phage-like particle, (iii) a general transducing agent (GTA), or a small, tailed bacteriophage-like particle, (iv) a Metamorphosis Associated Contractile structure (MACs), (v) a phage-derived product, or (vi) any combination of (i) to (v), as provided herein), are designed to and are capable of delivering a payload to a cell, e.g., a gut cell; for example, the payload can be a nucleic acid, e.g., a eukaryotic gene, optionally with appropriate promoters or enhancers and the like, for recombinant gene expression in a eukaryotic, e.g., a gut cell. For example, phages, prophages and the like as provided herein can deliver synthetic gene networks to correct metabolic deficiencies such as galactosemia. In alternative embodiments, phages, prophages and the like as provided herein deliver an iRNA or miRNA, or CRISPR cassettes, e.g., CRISPR or CRISPR/Cas9 nucleic acids, or a CRISPR/Cas9-gRNA complex, to target genes for modifying the physiology of a cell, e.g., to a cancer cell to treat the cancer, or add a gene to a cell, or replace a defective gene in a cell, or knockout a gene in a cell.
In alternative embodiments, compositions, products of manufacture and methods as provided herein (e.g., comprising the (i) a bacteriophage (“phage”), (ii) a prophage, a phagemid or a phage-like particle, (iii) a general transducing agent (GTA), or a small, tailed bacteriophage-like particle, (iv) a Metamorphosis Associated Contractile structure (MACs), (v) a phage-derived product, or (vi) any combination of (i) to (v), as provided herein), comprise a payload and can deliver the payload to a desired cell (e.g., a cell in the gut), e.g., deliver small compounds of therapeutics (e.g., drugs, nucleic acids) to cells. For example, in alternative embodiments, immunostimulatory compounds (e.g., vaccines, immunogens), or transcriptional activator proteins are delivered to cells to e.g., direct eukaryotic ribosomes to preferentially transcribe of phage-delivered genes, or anti-toxin compounds to prevent food-poisoning, and the like.
In alternative embodiments, compositions, products of manufacture and methods as provided herein (e.g., comprising the (i) a bacteriophage (“phage”), (ii) a prophage, a phagemid or a phage-like particle, (iii) a general transducing agent (GTA), or a small, tailed bacteriophage-like particle, (iv) a Metamorphosis Associated Contractile structure (MACs), (v) a phage-derived product, or (vi) any combination of (i) to (v), as provided herein), is delivered to and can traffic inside a eukaryotic cell (e.g., a gut cell) to target intracellular pathogens, parasites or agents, e.g., viral, bacterial, protozoan or fungal pathogens, e.g., Nocardia, Brucella, Francisella, Mycobacterium (e.g., Mycobacterium leprae and Mycobacterium tuberculosis), Legionella, Bartonella henselae, Francisella tularensis, Listeria monocytogenes, Salmonella enterica, Rhodococcus equi, Yersinia, Neisseria meningitidis Histoplasma capsulatum, Cryptococcus neoformans, Chlamydia, Rickettsia, Coxiella, Apicomplexa, Trpanosomatida or a Pneumocystis and the like, and kill or inactivate the intracellular pathogens or agents.
In alternative embodiments, for practicing methods, products of manufacture, and compositions as provided herein, provided are a particle, a nanoparticle, a liposome, a tablet, a pill, a capsule, a gel, a geltab, a liquid, a powder, a suspension, a syrup, an emulsion, a lotion, an ointment, an aerosol, a spray, a lozenge, an ophthalmic preparation, an aqueous or a sterile or an injectable solution, a patch (optionally a transdermal patch or a medicated adhesive patch), an implant, a dietary supplement, an ice cream, an ice, a yogurt, a cheese, an infant formula or infant dietary supplement, a pasteurized milk or milk product or milk-comprising product, or a liquid preparation embodiment or candies, lollies, drinks and the like, there can be added various preservatives, cryoprotectants and/or lyoprotectants, including e.g., various polysaccharides or sugars (such as sucrose, fructose, lactose, mannitol), glycerol, polyethylene glycol (PEG), trehalose, glycine, glucose, dextran and/or erythritol, comprising e.g., (i) bacteriophage (“phage”) (ii) prophage, the phagemid or phage-like particle, (iii) general transducing agent (GTA), or small, tailed bacteriophage-like particle, (iv) Metamorphosis Associated Contractile structure (MACs), (v) phage-derived product, or (vi) any combination of (i) to (v), as provided herein.
In alternative embodiments, cryoprotectants that can be used are ethylene glycol, 1,2-Propanediol, Methylcelliosolve, Dimethyl Formamide, or Dimethylsulphoxide Methanol. In alternative embodiments, the content of these cryoprotectants are between about 1% and about 50% but generally between about 5% and about 15% is adequate.
In alternative embodiments, a compound or composition for practicing methods, products of manufacture and compositions as provided herein is frozen and/or is freeze-dried, or spray dried, or lyophilized, using any method known in the art. For example, a method for freeze-drying bacteriophage can be used as described by Puapermpoonsiri et al., Int J. Pharm. 2010 Apr. 15; 389(1-2):168-75, who used sucrose or poly(ethylene glycol) 6000 to make bacteriophage-comprising freeze-dried cakes; or a method for making freeze-dried formulations of bacteriophage encapsulated in biodegradable microsphere, as described by Puapermpoonsiri et al., European J. Pharmaceutics and Biopharmaceutics, Vol. 72, Issue 1, 2009, Pgs 26-33; or methods for making stable bacteriophage compositions or matrices, as described e.g., by Murthy et al. WO2006047870 A1, or U.S. Pat. No. 8,309,077.
In alternative embodiments, there are different types of final products that can be manufactured. In alternative embodiments, a product or a formulation for practicing methods, products of manufacture and compositions as provided herein is a liquid. In alternative embodiments, a product or a formulation as provided herein is frozen and kept at e.g. minus 80 degrees for usage later given a cryoprotectant is added.
In alternative embodiments, biofilm disrupting compounds are added into a composition or formulation for practicing methods, products of manufacture and compositions as provided herein, provided are (e.g., a food, drink, nutraceutical, formulation, pharmaceutical or pharmaceutical preparation). In alternative embodiments, in practicing the methods as provided herein, biofilm disrupting compounds are administered before or during (co-administered), or co-formulated with (e.g., in a multi-laminated tablet or capsule), or separately formulated, as the administered composition or formulation as provided herein. In alternative embodiments, disrupting biofilms are used to separate from the colonic mucosa an adherent polysaccharide/DNA—containing layer, the so-called “biofilm”.
In alternative embodiments, other biofilm disrupting components or agents also can be used, e.g., enzymes such as a deoxyribonuclease (DNase), a N-acetylcysteine, an auranofin, alginate lyase, glycoside hydrolase dispersin B; Quorum-sensing inhibitors e.g., ribonucleic acid III inhibiting peptide, Salvadora persica extracts, Competence-stimulating peptide, Patulin and penicillic acid; peptides—cathelicidin-derived peptides, small lytic peptide, PTP-7 (a small lytic peptide, see e.g., Kharidia (2011) J. Microbiol. 49(4):663-8, Epub 2011 Sep. 2), Nitric oxide, neo-emulsions; ozone, lytic bacteriophages, lactoferrin, xylitol hydrogel, synthetic iron chelators, cranberry components, curcumin, silver nanoparticles, Acetyl-11-keto-β-boswellic acid (AKBA), barley coffee components, probiotics, sinefungin, S-adenosylmethionine, S-adenosyl-homocysteine, Delisea furanones, N-sulfonyl homoserine lactones and/or macrolide antibiotics or any combination thereof.
In alternative embodiments, biofilm disrupting components or agents are administered before and during the administration of a composition of this invention, e.g., as an antibacterial, in whatever format or formulation this may take place, for example, as a capsule.
In alternative embodiments, biofilm disrupting agents are added either before treatment and/or during and/or after treatment with a composition for practicing methods and compositions as provided herein. In alternative embodiments, biofilm disrupting agents are used singly or in combination.
In alternative embodiments, biofilm disrupting agents include particular enzymes and degrading substances including in N-acetylcysteine, deoxyribonuclease (DNase). Others would include Alginate, lyase and Glycoside hydrolase dispersin, Ribonucleic-acid-III inhibiting peptide (RIP), Salvadora persica extracts, Competence-stimulating peptide (CSP) Patulin (PAT) and penicillic acid (PA)/EDTA, Cathelicidin-derived peptides, Small lytic peptide, PTP-7, Nitric oxide, Chlorhexidine, Povidone-iodine (PI), Nanoemulsions, Lytic bacteriophages, Lactoferrin/xylitol hydrogel, Synthetic iron chelators, Cranberry components, Curcumin, Acetyl-11-keto-boswellic acid (AKBA). Barley coffee (BC) components, silver nanoparticles, azithromycin, clarithromycin, gentamicin, streptomycin and also Disodium EDTA. Ozone insufflations of the colon can also be used to disrupt the biofilm.
In alternative embodiments, a composition for practicing methods and compositions as provided herein (e.g., a particle, a nanoparticle, a liposome, a tablet, a pill, a capsule, a gel, a geltab, a liquid, a powder, a suspension, a syrup, an emulsion, a lotion, an ointment, an aerosol, a spray, a lozenge, an ophthalmic preparation, an aqueous or a sterile or an injectable solution, a patch (optionally a transdermal patch or a medicated adhesive patch), an implant, a dietary supplement, an ice cream, an ice, a yogurt, a cheese, an infant formula or infant dietary supplement, a pasteurized milk or milk product or milk-comprising product) can be further processed by, e.g., spray-drying or equivalent, e.g., spray-drying in an inert gas or freeze-drying under similar conditions, thus ending up with a powdered product.
In alternative embodiments, a composition as provided herein can be formulated for enteral or parenteral administration, e.g., to reach the systemic circulation, or for local delivery (e.g., for administration to skin, ears, teeth), as a topical for e.g., infections, as an inhalant, e.g., for inhalation of phages for the treatment of e.g., lung infections, as described e.g., by Ryan et al. J Pharm Pharmacol. 2011 October; 63(10):1253-64.
In alternative embodiments, a composition is manufactured, labelled or formulated as a liquid, a suspension, a spray, a gel, a geltab, a semisolid, a tablet, or sachet, a capsule, a lozenge, a chewable or suckable unit dosage form, or any pharmaceutically acceptable formulation or preparation. In alternative embodiments, a composition as provided herein is incorporated into a food or a drink (e.g., a yogurt, ice cream, smoothie), a candy, sweet or lolly, or a feed, a nutritional or a food or feed supplement (e.g., liquid, semisolid or solid), and the like.
For example, bacteriophage used to practice the invention can be encapsulated as described, e.g., by Murthy et al. in US 2012-0258175 A1. A composition as provided herein can be manufactured, labelled or formulated as an orally disintegrating tablet as described e.g., in U.S. Pat. App. Publication No. 20100297031. A composition as provided herein can be a polyol/thickened oil suspension as described in U.S. Pat. No. (USPN) 6,979,674; 6,245,740. A composition as provided herein can be encapsulated, e.g., encapsulated in a glassy matrix as described e.g., in U.S. Pat. App. Publication No. 20100289164; and U.S. Pat. No. 7,799,341. A composition as provided herein can be manufactured, labelled or formulated as an excipient particle, e.g., comprising a cellulosic material such as microcrystalline cellulose in intimate association with silicon dioxide, a disintegrant and a polyol, sugar or a polyol/sugar blend as described e.g., in U.S. Pat. App. Publication No. 20100285164. A composition as provided herein can be manufactured, labelled or formulated as an orally disintegrating tablet as described e.g., in U.S. Pat. App. Publication No. 20100278930. A composition as provided herein can be manufactured, labelled or formulated as a spherical particle, as described e.g., in U.S. Pat. App. Publication No. 20100247665, e.g., comprising a crystalline cellulose and/or powdered cellulose. A composition as provided herein can be manufactured, labelled or formulated as a rapidly disintegrating solid preparation useful e.g. as an orally-disintegrating solid preparation, as described e.g., in U.S. Pat. App. Publication No. 20100233278. A composition as provided herein can be manufactured, labelled or formulated as a solid preparation for oral application comprising a gum tragacanth and a polyphosphoric acid or salt thereof, as described e.g., in U.S. Pat. App. Publication No. 20100226866.
A composition as provided herein can be manufactured, labelled or formulated using a water soluble polyhydroxy compound, hydroxy carboxylic acid and/or polyhydroxy carboxylic acid, as described e.g., in U.S. Pat. App. Publication No. 20100222311. A composition as provided herein can be manufactured, labelled or formulated as a lozenge, or a chewable and suckable tablet or other unit dosage form, as described e.g., in U.S. Pat. App. Publication No. 20100184785.
A composition as provided herein can be manufactured, labelled or formulated in the form of an agglomerate, as described e.g., in U.S. Pat. App. Publication No. 20100178349. A composition as provided herein can be manufactured, labelled or formulated in the form of a gel or paste, as described e.g., in U.S. Pat. App. Publication No. 20060275223. A composition as provided herein can be manufactured, labelled or formulated in the form of a soft capsule, as described e.g., in U.S. Pat. No. 7,846,475, or 7,763,276.
The polyols used in compositions as provided herein can be micronized polyols, e.g., micronized polyols, e.g., as described e.g., in U.S. Pat. App. Publication No. 20100255307, e.g., having a particle size distribution (d50) of from 20 to 60 μm, and a flowability below or equal to 5 s/100 g, or below 5 s/100 g.
In practicing the invention, a wide variation of bacteriophage can be administered, for example, in some aspects, a smaller dosage can be administered because phage (i.e., bacteriophage) can replication in the host, i.e., in the individual to which a composition as provided herein is administered. In alternative embodiments, compositions as provided herein, including bacteriophages, phagemids or phage-like particles as provided herein, are formulated per dose, or per serving, or per unit dosage at, or at a total daily dose of between about 10(1) (or 101) and 10(20) plaque-forming units (PFUs), or between about 10(3) and 10(17) PFUs, or between about 10(5) and 10(12) PFUs, or between about 10(7) and 10(9) PFUs.
In alternative embodiments, provided are methods using compositions formulated for delayed or gradual enteric release comprising at least one active agent (e.g., a composition, a formulation or a pharmaceutical preparation as provided herein) formulated with a delayed release composition or formulation, coating or encapsulation. In alternative embodiments, formulations or pharmaceutical preparations as provided herein and used in methods provided herein are designed or formulated for delivery of active ingredient (e.g., a bacteriophage) into the distal small bowel and/or the colon. Thus, for this embodiment, it is important to allow the active ingredient to pass the areas of danger, e.g., stomach acid and pancreatic enzymes and bile, and reach undamaged to be viable in the distal small bowel and especially the colon. In alternative embodiments, a formulation or pharmaceutical preparation as provided herein is a liquid formulation, a microbiota-comprising formulation as provided herein and/or a frozen or a freeze-dried version thereof. In alternative embodiments, preferably for the encapsulated format, all are in powdered form.
In alternative embodiments, compositions as provided herein are formulated for delayed or gradual enteric release using cellulose acetate (CA) and polyethylene glycol (PEG), e.g., as described by Defang et al. (2005) Drug Develop. & Indust. Pharm. 31:677-685, who used CA and PEG with sodium carbonate in a wet granulation production process.
In alternative embodiments, compositions as provided herein are formulated for delayed or gradual enteric release using a hydroxypropylmethylcellulose (HPMC), a microcrystalline cellulose (MCC) and magnesium stearate, as described e.g., in Huang et al. (2004) European J. of Pharm. & Biopharm. 58: 607-614).
In alternative embodiments, compositions as provided herein are formulated for delayed or gradual enteric release using e.g., a poly(meth)acrylate, e.g. a methacrylic acid copolymer B, a methyl methacrylate and/or a methacrylic acid ester, a polyvinylpyrrolidone (PVP) or a PVP-K90 and a EUDRAGIT® RL PO™, as described e.g., in Kuksal et al. (2006) AAPS Pharm. 7(1), article 1, E1 to E9.
In alternative embodiments, compositions as provided herein are formulated for delayed or gradual enteric release as described in U.S. Pat. App. Pub. 20100239667. In alternative embodiments, the composition comprises a solid inner layer sandwiched between two outer layers. The solid inner layer can comprise a formulation or pharmaceutical preparation as provided herein and one or more disintegrants and/or exploding agents, one of more effervescent agents or a mixture. Each outer layer can comprise a substantially water soluble and/or crystalline polymer or a mixture of substantially water soluble and/or crystalline polymers, e.g., a polyglycol. These can be adjusted in an exemplary composition as provided herein to achieve delivery of the living components of an FMT distally down the bowel.
In alternative embodiments, compositions as provided herein are formulated for delayed or gradual enteric release as described in U.S. Pat. App. Pub. 20120183612, which describes stable pharmaceutical formulations comprising active agents in a non-swellable diffusion matrix. In alternative embodiments, a formulation or pharmaceutical preparation as provided herein is released from a matrix in a sustained, invariant and, if several active agents are present, independent manner and the matrix is determined with respect to its substantial release characteristics by ethylcellulose and at least one fatty alcohol to deliver bacteria distally.
In alternative embodiments, a formulation or pharmaceutical preparation as provided herein is formulated for delayed or gradual enteric release as described in U.S. Pat. No. 6,284,274, which describes a bilayer tablet containing an active agent (e.g., an opiate analgesic), a polyalkylene oxide, a polyvinylpyrrolidone and a lubricant in the first layer and a second osmotic push layer containing polyethylene oxide or carboxy-methylcellulose.
In alternative embodiments, a formulation or pharmaceutical preparation as provided herein is formulated for delayed or gradual enteric release as described in U.S. Pat. App. Pub. No. 20030092724, which describes sustained release dosage forms in which a nonopioid analgesic and opioid analgesic are combined in a sustained release layer and in an immediate release layer, sustained release formulations comprising microcrystalline cellulose, EUDRAGIT RSPO™, CAB-O-SIL™, sodium lauryl sulfate, povidone and magnesium stearate.
In alternative embodiments, a formulation or pharmaceutical preparation as provided herein is formulated for delayed or gradual enteric release as described in U.S. Pat. App. Pub. 20080299197, describing a multi-layered tablet for a triple combination release of active agents to an environment of use, e.g., in the GI tract. In alternative embodiments, a multi-layered tablet is used, and it can comprise two external drug-containing layers in stacked arrangement with respect to and on opposite sides of an oral dosage form that provides a triple combination release of at least one active agent. In one embodiment, the dosage form is an osmotic device, or a gastro-resistant coated core, or a matrix tablet, or a hard capsule. In these alternative embodiments, the external layers may contain biofilm dissolving agents and internal layers the living bacteria.
In alternative embodiments, a formulation or pharmaceutical preparation as provided herein is formulated as multiple layer tablet forms, e.g., where a first layer provides an immediate release of a formulation or pharmaceutical preparation as provided herein and a second layer provides a controlled-release of another (or the same) formulation or pharmaceutical preparation as provided herein, or another active agent, as described e.g., in U.S. Pat. No. 6,514,531 (disclosing a coated trilayer immediate/prolonged release tablet), U.S. Pat. No. 6,087,386 (disclosing a trilayer tablet), U.S. Pat. No. 5,213,807 (disclosing an oral trilayer tablet with a core comprising an active agent and an intermediate coating comprising a substantially impervious/impermeable material to the passage of the first active agent), and U.S. Pat. No. 6,926,907 (disclosing a trilayer tablet that separates a first active agent contained in a film coat from a core comprising a controlled-release second active agent formulated using excipients which control the drug release, the film coat can be an enteric coating configured to delay the release of the active agent until the dosage form reaches an environment where the pH is above four).
In alternative embodiments, a formulation or pharmaceutical preparation as provided herein is formulated for delayed or gradual enteric release as described in U.S. Pat. App. Pub. 20120064133, which describes a release-retarding matrix material such as: an acrylic polymer, a cellulose, a wax, a fatty acid, shellac, zein, hydrogenated vegetable oil, hydrogenated castor oil, polyvinylpyrrolidine, a vinyl acetate copolymer, a vinyl alcohol copolymer, polyethylene oxide, an acrylic acid and methacrylic acid copolymer, a methyl methacrylate copolymer, an ethoxyethyl methacrylate polymer, a cyanoethyl methacrylate polymer, an aminoalkyl methacrylate copolymer, a poly(acrylic acid), a poly(methacrylic acid), a methacylic acid alkylamide copolymer, a poly(methyl methacrylate), a poly(methacrylic acid anhydride), a methyl methacrylate polymer, a polymethacrylate, a poly(methyl methacrylate) copolymer, a polyacrylamide, an aminoalkyl methacrylate copolymer, a glycidyl methacrylate copolymer, a methyl cellulose, an ethylcellulose, a carboxymethylcellulose, a hydroxypropylmethylcellulose, a hydroxymethyl cellulose, a hydroxyethyl cellulose, a hydroxypropyl cellulose, a crosslinked sodium carboxymethylcellulose, a crosslinked hydroxypropylcellulose, a natural wax, a synthetic wax, a fatty alcohol, a fatty acid, a fatty acid ester, a fatty acid glyceride, a hydrogenated fat, a hydrocarbon wax, stearic acid, stearyl alcohol, beeswax, glycowax, castor wax, carnauba wax, a polylactic acid, polyglycolic acid, a co-polymer of lactic and glycolic acid, carboxymethyl starch, potassium methacrylate/divinylbenzene copolymer, crosslinked polyvinylpyrrolidone, polyvinylalcohols, polyvinylalcohol copolymers, polyethylene glycols, non-crosslinked polyvinylpyrrolidone, polyvinylacetates, polyvinylacetate copolymers or any combination. In alternative embodiments, spherical pellets are prepared using an extrusion spheronization technique, of which many are well known in the pharmaceutical art. The pellets can comprise one or more formulations or pharmaceutical preparations as provided herein, e.g., the liquid preparation embodiment.
In alternative embodiments, a formulation or pharmaceutical preparation as provided herein is formulated for delayed or gradual enteric release as described in U.S. Pat. App. Pub. 20110218216, which describes an extended release pharmaceutical composition for oral administration, and uses a hydrophilic polymer, a hydrophobic material and a hydrophobic polymer or a mixture thereof, with a microenvironment pH modifier. The hydrophobic polymer can be ethylcellulose, cellulose acetate, cellulose propionate, cellulose butyrate, methacrylic acid-acrylic acid copolymers or a mixture thereof. The hydrophilic polymer can be polyvinylpyrrolidone, hydroxypropylcellulose, methylcellulose, hydroxypropylmethyl cellulose, polyethylene oxide, acrylic acid copolymers or a mixture thereof. The hydrophobic material can be a hydrogenated vegetable oil, hydrogenated castor oil, carnauba wax, candellia wax, beeswax, paraffin wax, stearic acid, glyceryl behenate, cetyl alcohol, cetostearyl alcohol or and a mixture thereof. The microenvironment pH modifier can be an inorganic acid, an amino acid, an organic acid or a mixture thereof. Alternatively, the microenvironment pH modifier can be lauric acid, myristic acid, acetic acid, benzoic acid, palmitic acid, stearic acid, oxalic acid, malonic acid, succinic acid, adipic acid, sebacic acid, fumaric acid, maleic acid; glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, sodium dihydrogen citrate, gluconic acid, a salicylic acid, tosylic acid, mesylic acid or malic acid or a mixture thereof.
In alternative embodiments, a formulation or pharmaceutical preparation as provided herein is a powder that can be included into a tablet or a suppository. In alternative embodiments, a formulation or pharmaceutical preparation as provided herein can be a ‘powder for reconstitution’ as a liquid to be drunk or otherwise administered. In alternative embodiments, a formulation or pharmaceutical preparation as provided herein is micro-encapsulated, formed into tablets and/or placed into capsules, especially enteric-coated capsules.
In alternative embodiments, in practicing the methods as provided herein, buffers or antacids are administered before or during (co-administered), or co-formulated with a composition or formulation as provided herein. For example, in alternative embodiments, a composition or formulation as provided herein and a buffer or antacid are co-formulated, e.g., as multiple layer tablet form or as a multi-laminated tablet or capsule. In alternative embodiments of methods as provided herein, buffers or antacids are separately formulated. In alternative embodiments, the antacid, buffer or buffering agent is administered (optionally before, during or after, or before and during, administration) to raise the pH of the stomach in the individual to between about 2.5 and 7, or between about 3 and 6.5, or to about 5.0, 5.5, 6.0, 6.5, 6.8 or 7.0 (optionally these pH values reached before, during or after, or before and during, administration). In alternative embodiments, the buffer or a buffering agent or the pharmaceutically acceptable excipient comprises an inorganic salt, a citric acid, a sodium chloride, a potassium chloride, a sodium sulfate, a potassium nitrate, a sodium phosphate monobasic, a sodium phosphate dibasic or combinations thereof. In alternative embodiments, the antacid comprises a calcium carbonate, a magnesium hydroxide, a magnesium oxide, a magnesium carbonate, an aluminum hydroxide, a sodium bicarbonate or a dihydroxyaluminum sodium carbonate.
In alternative embodiments, a formulation or pharmaceutical preparation as provided herein, or used in a method provided herein, is incorporated into a food, a feed, a candy (e.g., a lollypop or a lozenge) a drink, a nutritional or a food or feed supplement (e.g., liquid, semisolid or solid), and the like, as described e.g., in U.S. Pat. App. Publication No. 20100178413. In one embodiment, a formulation or pharmaceutical preparation as provided herein is incorporated into (manufactured as) a beverage as described e.g., in U.S. Pat. No. 7,815,956. For example, a composition as provided herein is incorporated into a yogurt, an ice cream, a milk or milkshake, a “frosty”, “snow-cone”, or other ice-based mix, and the like.
In alternative embodiments, a formulation or pharmaceutical preparation as provided herein is a freeze-dried powder form added to a food, e.g., a yogurt, an ice cream, a milk or milkshake, a “frosty”, “snow-cone”, or other ice-based mix, and the like. In one form of this invention it can be kept in a lid-storage (e.g., of a yogurt or ice cream) such that when it is twisted the powder falls into the product or formulation (e.g., yoghurt or ice cream) and then it can be stirred so as not to have the powder ferment ‘standing on the shelf’. Various flavourings can be added. In alternative embodiments, this is particularly important for administration of a composition as provided herein, e.g., a wild type microbiota or a cultured bacteria, to a very young individual and/or a patient with autism or related disease or condition.
In alternative embodiments, these exemplary products are important when administered to children or babies who may have acquired various pathogenic or abnormal bacteria, e.g., E. coli, Clostridia or Disulfovibrio, e.g., as in autism.
Compositions as provided herein and used to practice methods as provided herein (e.g., a product of manufacture, food, drink, nutraceutical, formulation, pharmaceutical or pharmaceutical preparation), including preparations, formulations and/or kits, comprise combinations of ingredients, as described herein. In alternative embodiments, these combinations can be mixed and administered together, or alternatively, they can be an individual member of a packaged combination of ingredients, e.g., as manufactured in a separate package, kit or container; or, where all or a subset of the combinations of ingredients are manufactured in a separate package or container. In alternative aspects, the package, kit or container comprises a blister package, a clamshell, a tray, a shrink wrap and the like.
In one aspect, the package, kit or container comprises a “blister package” (also called a blister pack, or bubble pack). In one aspect, the blister package is made up of two separate elements: a transparent plastic cavity shaped to the product and its blister board backing. These two elements are then joined together with a heat sealing process which allows the product to be hung or displayed. Exemplary types of “blister packages” include: Face seal blister packages, gang run blister packages, mock blister packages, interactive blister packages, slide blister packages.
Blister packs, clamshells or trays are forms of packaging used for goods; thus, the invention provides for blister packs, clamshells or trays comprising a composition (e.g., a (the multi-ingredient combination of drugs as provided herein) combination of active ingredients) as provided herein. Blister packs, clamshells or trays can be designed to be non-reclosable, so consumers can tell if a package has already opened. They are used to package for sale goods where product tampering is a consideration, such as the pharmaceuticals as provided herein. In one aspect, a blister pack as provided herein comprises a moulded PVC base, with raised areas (the “blisters”) to contain the tablets, pills, etc. comprising the combinations as provided herein, covered by a foil laminate. Tablets, pills, etc. are removed from the pack either by peeling the foil back or by pushing the blister to force the tablet to break the foil. In one aspect, a specialized form of a blister pack is a strip pack. In one aspect, in the United Kingdom, blister packs adhere to British Standard 8404.
In one embodiment, provided are methods of packaging where the compositions comprising combinations of ingredients as provided herein are contained in-between a card and a clear PVC. The PVC can be transparent so the item (pill, tablet, geltab, etc.) can be seen and examined easily; and in one aspect, can be vacuum-formed around a mould so it can contain the item snugly and have room to be opened upon purchase. In one aspect, the card is brightly colored and designed depending on the item (pill, tablet, geltab, etc.) inside, and the PVC is affixed to the card using pre-formed tabs where the adhesive is placed. The adhesive can be strong enough so that the pack may hang on a peg, but weak enough so that this way one can tear open the join and access the item. Sometimes with large items or multiple enclosed pills, tablets, geltabs, etc., the card has a perforated window for access. In one aspect, more secure blister packs, e.g., for items such as pills, tablets, geltabs, etc. as provided herein are used, and they can comprise of two vacuum-formed PVC sheets meshed together at the edges, with the informative card inside. These can be hard to open by hand, so a pair of scissors or a sharp knife may be required to open.
In one aspect, blister packaging comprises at least two or three or more components (e.g., is a multi-ingredient combination as provided herein): a thermoformed “blister” which houses multi-ingredient combination as provided herein, and then a “blister card” that is a printed card with an adhesive coating on the front surface. During the assembly process, the blister component, which is most commonly made out of PVC, is attached to the blister card using a blister machine. This machine introduces heat to the flange area of the blister which activates the glue on the card in that specific area and ultimately secures the PVG blister to the printed blister card. The thermoformed PVG blister and the printed blister card can be as small or as large as you would like, but there are limitations and cost considerations in going to an oversized blister card. Conventional blister packs can also be sealed (e.g., using an AERGO 8 DUO™, SCA Consumer Packaging, Inc., DeKalb Ill.) using regular heat seal tooling. This alternative aspect, using heat seal tooling, can seal common types of thermoformed packaging.
In alternative embodiments, combinations of ingredients of compositions as provided herein or used to practice methods provided herein, or combinations of ingredients for practicing methods as provided herein, can be packaged alone or in combinations, e.g., as “blister packages” or as a plurality of packettes, including as lidded blister packages, lidded blister or blister card or packets or packettes, or a shrink wrap.
In alternative embodiments, laminated aluminium foil blister packs are used, e.g., for the preparation of drugs designed to dissolve immediately in the mouth of a patient. This exemplary process comprises having the drug combinations as provided herein prepared as an aqueous solution(s) which are dispensed (e.g., by measured dose) into an aluminium (e.g., alufoil) laminated tray portion of a blister pack. This tray is then freeze-dried to form tablets which take the shape of the blister pockets. The alufoil laminate of both the tray and lid fully protects any highly hygroscopic and/or sensitive individual doses. In one aspect, the pack incorporates a child-proof peel open security laminate. In one aspect, the system gives tablets an identification mark by embossing a design into the alufoil pocket that is taken up by the tablets when they change from aqueous to solid state. In one aspect, individual ‘push-through’ blister packs/packettes are used, e.g., using hard temper aluminium (e.g., alufoil) lidding material. In one aspect, hermetically-sealed high barrier aluminium (e.g., alufoil) laminates are used. In one aspect, any as provided herein's products of manufacture, including kits or blister packs, use foil laminations and strip packs, stick packs, sachets and pouches, peelable and non-peelable laminations combining foil, paper, and film for high barrier packaging.
In alternative embodiments, any as provided herein's multi-ingredient combinations or products of manufacture, including kits or blister packs, include memory aids to help remind patients when and how to take the drug. This safeguards the drug's efficacy by protecting each tablet, geltab or pill until it's taken; gives the product or kit portability, makes it easy to take a dose anytime or anywhere.
The invention will be further described with reference to the following examples; however, it is to be understood that the invention is not limited to such examples.
This example provides data evidence demonstrating that a (i) bacteriophage (“phage”) (ii) prophage, the phagemid or phage-like particle, (iii) general transducing agent (GTA), or small, tailed bacteriophage-like particle, (iv) Metamorphosis Associated Contractile structure (MACs), (v) phage-derived product, or (vi) any combination of (i) to (v), as provided herein can effectively and in sufficient amounts enter an animal, e.g., a mammal, e.g., an individual in need thereof, to deliver sufficient amounts of, and optionally target to a specific cell, tissue or organ, the (i) bacteriophage (“phage”) (ii) prophage, the phagemid or phage-like particle, (iii) general transducing agent (GTA), or small, tailed bacteriophage-like particle, (iv) Metamorphosis Associated Contractile structure (MACs), (v) phage-derived product, or (vi) any combination of (i) to (v), into the blood stream or lymphatic system of the animal, e.g., a mammal, or deliver the (i) bacteriophage (“phage”) (ii) prophage, the phagemid or phage-like particle, (iii) general transducing agent (GTA), or small, tailed bacteriophage-like particle, (iv) Metamorphosis Associated Contractile structure (MACs), (v) phage-derived product, or (vi) any combination of (i) to (v), to a tissue or organ of the animal, e.g., a mammal, in vivo, for e.g., treating, ameliorating and/or preventing an infection, disease or condition in an individual in need thereof, where the infection, disease or condition is outside of the gut or gastrointestinal (GI) tract.
Here we show that bacteriophage transcytosis across diverse epithelial cell layers was both irregular but repeatable, with a strong apical-to-basolateral membrane directionality. Bacteriophages transit through the Golgi and accessed microsomal fractions of the cell, suggesting free uptake by endocytosis as the mechanism to access the body. Using experimental data, we estimate that thirty-billion bacteriophage particles are transcytosed by the average human body every day, with comparable ingress via a ‘leaky gut’ requiring significant intestinal damage. The transcytosis of bacteriophage into the body is a natural and ubiquitous process that may have important implications, including vertical transmission of gut phages and immunostimulatory effects on the body.
Escherichia coli B strain HER 1024 was grown in LB (10 g tryptone, 5 g yeast extract, 10 g NaCl, in 1 L dH2O) at 37° C. shaking overnight and used to propagate and quantify bacteriophages T4, T3, T5 and T7. Bacillus subtilis 168WT was grown in TY broth (10 g tryptone, 5 g yeast extract, 5 g NaCl, 10 mM MgSO4, 100 μM MnSO4, in 1 L dH2O) at 37° C. shaking for 6-8 hrs and used to propagate and quantify bacteriophages SP01 and SPP1. Salmonella typhimurium LT2 was grown in LB at 37° C. shaking overnight and used to propagate and quantify bacteriophages P22. All phage lysates were purified and cleaned of bacterial endotoxins according to the Phage-On-Tap protocol (1).
All tissue culture cells lines were grown at 37° C. and 5% CO2 and supplemented with 1% Penicillin/Streptomycin (Mediatech, Inc., Tewksbury, Mass.). MDCK.2 cells were grown in Eagles's Minimal Essential Media with 10% Fetal Bovine Serum (FBS), T84 cells were grown in Ham's F12 medium and Dulbecco's modified Eagle's medium with 2.5 mM L-glutamine with 10% FBS, CaCo2 cells were grown in Eagles's Minimal Essential Media with 10% FBS, A549 and Huh7 cells were grown in F-12K medium with 10% FBS, hBMec cells were grown in RPMI medi with 10% nuSerum (Corning, N.Y.) and 1% NEAA (GIBCO, Walktham, Mass.).
TRANSWELL® PET 12 well plates with 0.4 μm pore size (Corning, N.Y.) were used for all transcytosis assays. All cells were seeded at a density of 0.5-1×106 cells per well and allowed to grow to confluency (3-5 days). For apical-to-basal transcytosis the apical wells were incubated in Hanks Buffered Salt Solution (HBSS) at pH 6.0 and basal cells in HBSS at pH 7.4 for two hours to mimic pH-dependent uptake (2). For basal-to-apical transcytosis the buffers were switched. Bacteriophages were applied with the HBBS pH 6.0 buffer, incubated with cells for two hours and phage from both apical and basal cell layers quantified by plating with bacterial host.
Cell layer confluency of all transwell experiments were measured in three separate ways to ensure phage transcytosis across the cell layer, rather than by paracellular transport. Firstly, a visual inspection using a phase-contrast microscope. Secondly, transepithelial resistances (TER) of all cell lines were measured (World Precision Instruments, Sarasota Fla.), with the acceptable range of measurements between 150-200 Ω*cm2. TER measurements were taken before and after all transcytosis experiments to ensure cell confluency and polarization had been reached and maintained. Finally, 250 μL of HBSS buffer with 25 μL of Evans blue dye was added to the apical chambers of all transwells post-assay and incubated with cells at 37° C. for 2 hrs. Basal chambers were collected and absorbance was measured (620 nm) using a spectrophotometer and was compared against an Evans blue dilution curve (Fig S1). The presence of dye in the basal chamber was indicative of a non-confluent cell layer and data from these wells were discarded.
MDCK cells grown to confluence were incubated with T4 phages for either 5 minutes or 18 hours. Cell layers were then extensively washed with DPBS and subjected to microsomal fractionation (3), using the Lysosomal Enrichment™ kit for Tissue and Cultured Cells according to manufacturer's instructions (Thermo-Fisher). Briefly, approximately 200 mg of cells were harvested with trypsin and centrifuged for 2 min at 850×g. Lysosome enrichment reagent A containing a protease inhibitor cocktail (CalBioChem) was added to pelleted cells and subjected to a 2 min incubation on ice. After incubation, cells were then sonicated 15 times to lyse the cells, followed by addition of Lysosome enrichment reagent B containing a protease inhibitor. Cells were then centrifuged for 10 min at 500×g at 4° C. The supernatant was then collected and the final concentration was altered to 15% with OPTIPREP CELL SEPARATION MEDIA™. Samples were then loaded on a discontinuous OPTIPREP™ gradient from 60%. 30%, 27%, 23%. 20% to 17% om a 13.2 mL ultracentrifugation tube (Beckman-coulter) and centrifuged in a SW 41 Ti rotor at 145,000×g for 2 hours at 4° C. After ultracentrifugation, the lysosomal fraction was isolated from the top of the gradient, and all other microsomal fractions were isolated (Fig S2). All microsomal fractions were washed using two volumes of DPBS in a microcentrifuge tube at 17,000×g for 30 min at 4° C. Microsomal pellets were then washed with DPBS and centrifuged again at 17,000×g for 30 min at 4° C. Pellets were lysed with 0.1 volumes chloroform for 10 min, followed by centrifugation at 17,000×g for 5 min. Supernatants were then plated with bacterial hosts and phages quantified.
Graphing and statistical analyses were performed using GRAPHPAD PRISM 7™ (GraphPad Prism; GraphPad Software). Individual data points, medians and standard deviations were reported where possible (4). Both non-parametric and parametric statistical analyses were performed, although most data did not pass a normality test.
Phage transcytosis rate. We calculate the transcytosis rate per unit time, surface area (wall), and concentration of T4 phages and T84 gut epithelial cells. In a first approximation, this leads to:
where ϕlo is the basal concentration of phages, Vlo is the volume of the lower compartment, Slo is the surface area of a single Transwell, ϕup is the apical concentration of phages, and tin is the time of incubation. The first term (ϕloVlo,Slo=σ) represents the number of phages that accomplished transcytosis per unit area of the Transwell. This number depends on how many phages contact the epithelial cells on the apical side. Thus, we divide by the apical concentration of phages (ϕup). This assumes that the transcytosis mechanism is independent on the number of phages contacting the cell and being transported at a given time. The duration of the experiment will also impact the number of phages that are counted in the basal part of the cell. Thus, we divide by the time of incubation (tin), which assumes that the rate of the transcytosis mechanism is approximately constant during the time scale of the experiment. This leads to a transcytosis rate, rtr, of 0.166 μm/h.
Number of transcytosed phages in humans. We estimate the number of phages that are being transcytosed in one day (24 hours) in the average human body. To calculate this number we combine the experimentally derived transcytosis rate with physiological data. Using the model in Eq. (S1), the number of phages transcytosed in humans per day is:
In this equation, we multiply the transcytosis rate (rtr) by the surface area of the large intestine (Sli) times the concentration of phages in the intestine (Φli/Vli) times 24 hours (1 day). In this way, we estimate that there are seven billion phages that penetrate the human body per day using the transcytosis route.
Mucus factor. We then assume a 4.4-fold increase of phage numbers associated with mucosal surfaces in the large intestine, giving a total of
Φtr,mday≈4.4×Φtrday≈30.97·109 phages/day Eq. (S3)
Thus, there are approximately thirty-one billion phages transcytosed by the human body per day. The constants used in this model are reported in Table S5 (physiological parameters in the large intestine), Table S6 (parameters of the transcytosis experiment), and Table S7 (model results).
Transcytosis in MCDK cells. The results above were based on the experiments done in T84 cells. Here we calculate the factor required to extend the results to Madin-Darby canine kidney cells (MCDK). In T84 cells, when 4.7×107 phage ml−1 are applied to the apical side and 7.9×103 phage ml−1 were recovered in the basal side. This leads to a raw transcytosis ratio of 1.48×101. In MCDK cells, 3.2×107 phage ml−1 were applied to the apical side, and 1.9×104 phage ml−1 were recovered in the basal side, that is, a raw transcytosis ratio of 2.9×10−4. Thus, the transcytosis ratio is 1.96 times higher in MCDK cells than in T84 cells. To estimate the transcytosis in MCDK we multiply our modelled results of T84 by ƒ≈1.96, giving a transcytosis rate, rtr, of 0.325 μm/h.
We assume phages can bypass confluent epithelial layers at sites of inflammation caused by cellular damage and punctured vasculature. Here we introduce a mathematical model to estimate the flux of phages penetrating the body using this route. The constants used in and the values obtained from the model are summarized in Table S8.
Leaky-gut model upper bound limit. In a first approximation, we consider that every damaged region in the gut is equivalent to removing an entire epithelial cell, thus opening a channel 40 um long (Table S5), and we assume that the channel is filled with the same fluid as the gut surface. This allows phages to diffuse from the gut to the lymphatic and blood circulatory system. To obtain an upper bound limit to the number of phages penetrating the body by this mechanism, we neglect entropic effects associated to the section or number of channels, that is, we consider that multiple punctured points or a single hole with the same effective section lead to the same leaking. Under this assumption, phages will have the same diffusion constant both in the gut and in the channel. In the upper bound limit, we consider that phages diffuse as if they were in water at the body temperature (37° C.). The diffusion constant, Dw, as stated in the Einstein-Smoluchowski equation, is the ratio of the thermal energy, kBT, and the friction coefficient of phages in water, γw, as given by Eq. (S4).
Here kB is the Boltzmann constant. The friction constant is obtained by applying the Stokes-Einstein relation as shown in the denominator of the third term in Eq. (S4), where Rϕ is the effective radius of the phage; most phages are quasi-spherical and have a similar size to lambda phage, so we assume an effective radius of Rϕ≈30 nm. The viscosity of water at body temperature is vw=0.6913 mPa s I (8). This leads to an approximate phage diffusion constant of 11 μm2/s, that is, phages cover an effective region of radius √11≈3.3 μm per second.
The flux of phages penetrating the body, Jw, is proportional to the diffusion coefficient, Dw, and the gradient of phage concentration in the transport channel as given by the Fick's law:
To calculate the flux, we need to determine the concentration profile of phages across the channel. This profile is determined by the diffusion equation:
We consider that the side of the channel in the gut provides a constant supply of phages, ϕ(0)=ϕ0, while in the other side of the channel (blood stream or lymphatic system) the phages do not accumulate, ϕ(H)=0, where H is the “length” of the channel (height in Table S5). This will eventually lead to a stable concentration profile that does not change in time, i.e., it is stationary:
In this situation, the concentration of phages is determined by the Laplace equation—right term in Eq. (S7). Integrating this differential equation and applying the boundary conditions, ϕ(0) and ϕ(H), give us the concentration profile:
Applying this profile into the Fick's law equation, Eq. (S4), we obtain the general expression of the flux of phages in the leaky-gut model:
Applying the value of the phage diffusion in water at body temperature, Dw (Eq. S3), the concentration of phage in the large intestine, ϕ0=ϕli (Table S5), and the height of the epithelial cell, H (Table S5), we obtain a flux of phages of 1.4×10−3 per unit area (μm2) and time (s).
How does this flux compare to the number of phages penetrating the body by the transcytosed mechanism? To answer this, we estimate the effective section of the channel (or number of epithelial cells removed) necessary to lead to the same number of phages per day obtained in the gut in Eq. (S3). This condition is expressed as:
J
w
τS*
w=Φtr,mday Eq. (S10)
That is, the flux times the time (τ=24 h) times the damaged surface (S*w) equates the number of phages transcytosed per day in the long intestine (Eq. S3). This leads to:
Taking into account the section of an epithelial cell, Sec (Table S6), we obtain that the number of damaged epithelial cells:
Thus, the leaky-gut mechanism requires more than ten million epithelial cells to be removed to reach a similar number of phages penetrating the body compared to the phage transcytosis mechanism. Notice that the flux in this case was an upper limit, so the number of damaged cells is a lower limit. If we introduce more realistic details in the model (e.g., wall effects, non-homogeneous flux across the section, entropic cost to enter the channel), this number would increase considerably.
T4 phage transcytosis across eukaryotic cells. The directional transcytosis of T4 phage particles across eukaryotic cells was measured using Transwell inserts seeded with Madin-Darby canine kidney cells (MDCK) that were grown to confluence (
To determine whether these observations with the MDCK cell line could be extended to other human associated tissues, we examined cell lines derived from distinct organs, and which form confluent monolayers, including those from the gut (T84 and CaCo2), lung (A549), liver (Huh7) and brain (hBMec) (
Functionality of phage transcytosis. The ingress of phages throughout the body has been previously described (4-12). However, there have been no quantitative measurements of the rate, dose or generality of the phenomenon. Using T4 phages and MDCK cells, the rate of apical-to-basal transcytosis was recorded over a two-hour period (
Phage preparations are often contaminated by host bacteria macromolecules, with the major pyrogen being lipopolysaccharide (endotoxin) (32). Endotoxin is known to elicit a wide range of pathophysiological effects in the body, stimulating cellular and immune responses (33). To investigate whether residual endotoxins were triggering phage transcytosis, we compared a T4 phage stocks before and after removal of endotoxins (34). The removal of endotoxins produced no significant change in apical-to-basal transcytosis of T4 phage (
The generality of phage transcytosis was next tested using diverse phages across the order of Caudovirales, encompassing phages from; the three major morphotypes (Myoviridae, Siphoviridae, Podovirdae), Gram positive and negative bacterial hosts, and phages originating from soil and intestinal reservoirs. All phages tested elicited strong apical-to-basal transcytosis (
Permeation of phages throughout the eukaryotic cell. The mechanism of phage access to Eukaryotic cells remains ambiguous (22, 24, 29). To identify this mechanism, we applied chemical inhibitors known to arrest steps along the transcytotic pathway in MDCK cells prior to application of T4 phages. Inhibition of phage transcytosis was reported as the percentage of phages transcytosed across inhibitor-treated cells, compared to cells treated with a solvent control (
The proportion of fluorescence-positive cells treated with BFA and labeled T4 phages was 9.98% (n=1008, 9.98%±0.78%, mean±s.e.)
Subcellular fractionations were performed to assess T4 phage localization within MDCK cells. Cells were treated with T4 phage for either 5 minutes or 18 hours, washed, fractionated and the vesicular and cytoplasmic cellular components collected. Cells treated for 5 minutes showed no detectable phages in any subcellular fractions, likely due to the insufficient incubation time (
T4 phage were fluorescently labeled and visualized within MDCK cells (
The proportion of fluorescence-positive cells treated with labeled T4 phages was 10.54% (n=2961, 10.54%±0.81%, mean±s.e.) and 1.7% (n=1650, 1.7%±0.5%, mean±s.e.) as analyzed by epifluorescence microscopy and flow-cytometry, respectively. This is in contrast to our prior observation that 0.1% of total phage applied were transcytosed (
Estimates of phage ingress to the human body. Within the human body the largest aggregation of phages resides within the gut (17, 37). Although the concentration of bacteria within the human gut (averages 9.17×1010 per gram of feces) has been well documented (38, 39), direct quantification of phages is comparatively lacking. Based on three literature references utilizing direct counts and DNA yield, we estimate 5.09×109 phage per gram of feces (2, 40, 41), yielding 2.09×1012 phage within the colon of an average human (39). Using our experimentally derived rate of phage transcytosis across T84 gut epithelial cells, and assuming a 4.4-fold increased concentration of phage in mucosal surfaces (42), we estimate that the average human body transcytoses 3.09×1010 phages per day. Finally, we contrast this estimate with a competing mechanism of access to the body via a ‘leaky gut’. In this model free phages are allowed to bypass confluent epithelial layers at sites of damage and inflammation, gaining access to the body directly (19, 20). To achieve similar phage ingress to our transcytosis model, we estimate this requires lesions of approximately 256 mm2 within the gastrointestinal tract, or the removal of 10.24×106 epithelial cells. This amount of intestinal damage would likely result in significant inflammation of the gut and is in contrast to the detection of phages in asymptomatic humans (8-12).
We selected a SYBR-gold positive target cell using confocal microscopy (
To address the lack of fluorescence and TEM ultrastructure correlation, we performed a time-series experiment using dual-fluorescence labeled phages that were incubated with MDCK cells for either 30 min or two hrs (
Permeation and inhibition of phages throughout the eukaryotic cell: Subcellular fractionation was performed to assess intracellular T4 phage dispersal within MDCK and A549 cells. To ensure maximal uptake and penetration of phages throughout the subcellular structure, cells were incubated with phages for 18 hrs, extensively washed, fractionated and the vesicular and cytoplasmic cellular components collected. Vesicular fractions were then split, with half of the fraction lysed using chloroform and the total number of phage quantified by plating with their bacterial host, and the remaining fraction protein precipitated and analyzed by immunoblotting using Golgi and endoplasmic reticulum markers (
Before this invention, the mechanism of phage transcytosis across eukaryotic cells remained ambiguous, see e.g., Duerkop et al., 2013, Resident viruses and their interactions with the immune system, Nat Immunol 14:654-9; Merril, et al., 1996, Long-circulating bacteriophage as antibacterial agents, Proc Natl Acad Sci 93:3188-3192; Aronow, et al., 1964, Electron microscopy of in vitro endocytosis of T2 phage by cells from rabbit perioneal exudate, J Exp Med 120. We applied chemical inhibitors known to arrest steps along the transcytotic pathway to MDCK cells 18 hrs prior to application of T4 phages. Inhibition of phage transcytosis was reported as the percentage of phages transcytosed across inhibitor-treated cells compared to cells treated with a solvent control (
Estimates of phage ingress to the human body: Within the human body the largest aggregation of phages resides within the gut (20, 43). Although the concentration of bacteria within the human gut (averages 9.17×1010 per gram of feces) has been well documented (44, 45), direct quantification of phages is comparatively lacking. Based on three literature references (Clokie et al., 2011. Phages in nature. Bacteriophage 1:31-45; Kim et al., 2011, Diversity and abundance of single-stranded DNA viruses in human feces. Appl Environ Microbiol 77:8062-70; Reyes et al., 2013, Gnotobiotic mouse model of phage-bacterial host dynamics in the human gut, Proc Natl Acad Sci USA 110:20236-41) utilizing direct counts and DNA yield, we estimate 5.09×101 phage particles per gram of feces, yielding 2.09×1012 phage particles within the colon of an average human. Using our experimentally derived rate of phage transcytosis across T84 gut epithelial cells, and assuming a 4.4-fold increased concentration of phage in mucosal surfaces, we estimate that the average human body transcytoses 3.1×1010 phages per day.
Finally, we contrast this estimate with a competing mechanism of access to the body via a ‘leaky gut’. In this model, free phages are allowed to bypass confluent epithelial layers at sites of damage and inflammation, gaining access to the body directly. To achieve similar phage ingress to our transcytosis model, we estimate this requires lesions of approximately 256 mm2 within the gastrointestinal tract, or the removal of 10.24×106 epithelial cells. This amount of intestinal damage would likely result in significant inflammation of the gut and is in contrast to the detection of phages within the blood and serum of asymptomatic humans.
Cellular investigations showed phages were capable of accessing all subcellular fractions of the eukaryotic cell (
The observation of microorganisms present within the blood and body is long-standing. Numerous mechanisms for bacterial uptake and invasion, and their subsequent effects have been identified. Comparatively, the mechanisms for bacteriophage uptake and potential effects on the body remain unaddressed. This is partly due to an underappreciated notion for phage-Eukaryote interactions, but also stems from the irregular and low rate of phage transcytosis, and the inherent difficulties associated with the molecular identification of low abundance phage (45). The identification of an intra-body ‘phageome’ presents a significant challenge (46), yet the potential implications for health and disease demands additional research into this underexplored area.
The transcytosis of bacteriophage across epithelial cells (
If the human body is perpetually absorbing phage, what might be the intended function? The major reservoir of phage in the body is observed in the gastrointestinal tract. Over the lifetime of a human these gut phage co-evolved with the microbiome, and represent the most genetic diversity and “biological dark matter” in the body (47, 48). At the simplest level, the presence of a low-level but continuous stream of phage originating from the gut and disseminating through the blood, lymph and organs, may provide the host with a system-wide antimicrobial against the intrusion of any opportunistic gut microbe. Their dissemination may have additional roles in cellular disease, cancer recognition and even the vertical transmission of adapted gut phage populations from mother to infant through breast milk (49-51).
At the same time, this continuous and low-level stream of phage represents a persistent influx of foreign and thus immunogenic particles throughout the body. Phages capacity to stimulate humoral responses and induce anti-phage antibodies is dependent on both their route of administration and dosage (7, 52). Transcytosed phage are continuously dosed to the body at relatively low levels, with a diversity that reflects current gut conditions and that lack costimulatory signals such as endotoxins (32). As such the immunostimulatory effects of transcytosed phages on the body are largely unknown. Nonetheless, their presence within the body could provide long-term immunological tolerance through interactions with regulatory T cell populations (53). Alternatively, aberrant transcytosis may contribute to enhanced immune responses, allergic reactions and inflammatory diseases (54).
Perhaps the greatest potential function of transcytosed gut phages is the utilization of their astounding genetic diversity by the body directly. Previous work using recombinant T4 phages has already documented the delivery and expression of single or multiple genes to Eukaryotic cells both in vitro and in vivo (25). The transcytosis of diverse phages reported here provides a mechanism to traverse the Eukaryotic cell. The subsequent intracellular dissemination of these phages and their genetic material provides a means to directly affect the Eukaryotic cell. Nominally this allows for horizontal gene transfer between phages and Eukaryotes (55) and the direct uptake and expression of phage genetic material within the body, potentially representing an unexplored third external genome (56). Studies provided herein demonstrate that the transcytosis of bacteriophage into the body across polarized epithelial cells is a naturally occurring and ubiquitous process, and for the first time validates the use and application of phages in an in vivo biomedical setting.
5 × 103
6 × 106
3 × 102
8 × 106
2 × 104
.48 ± 2 × 104
1 ± .19 × 105
2 ± .64 × 103
8 ± 6.2 × 103
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
This U.S. Utility Patent Application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application Ser. No. (USSN) 62/444,269, filed Jan. 9, 2017. The aforementioned application is expressly incorporated herein by reference in its entirety and for all purposes.
This invention was made with government support under grant no. DK53056. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US18/12983 | 1/9/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62444269 | Jan 2017 | US |
